The invention relates to novel fungal growth media and methods for the in vitro aseptic mass production of endomycorrhizal fungal propagules, mainly spores, using transformed root organ culture.
Vesicular-arbuscular mycorrhizal (VAM) fungi form beneficial symbiotic associations with roots of a wide range of plants (Harley and Smith, 1983). The term xe2x80x9cmycorrhiza,xe2x80x9d which literally means xe2x80x9cfungus root,xe2x80x9d was first used in 1885 by A. B. Frank to describe the intimate associations between fungal hyphae and the roots of forest trees. The hyphae of VA mycorrhizal fungi penetrate and form arbuscules within root cortical cells and intercellular vesicles, but the external hyphae extend further into the soil for mineral nutrient uptake. A simple interpretation of mycorrhizal symbiosis is that the plant supplies carbon compounds to increase fungal growth and the plant benefits by having its root system effectively extended. The surface area of fungal hyphae can be much greater than that of the plant roots (Smith and Gianinazzi-Pearson; 1992, Smith and Read, 1997).
The importance of VA mycorrhizae in the growth and nutrition of plants has recently been reviewed (Gianinazzi and Schhepp, 1994; Smith and Read, 1997; Harrison, 1999). On soils low in available phosphate, the improved growth observed in VA mycorrhizal plants compared with uninoculated controls is thought to be due largely to an improved supply of phosphate. The external hyphae in soil absorbs non-mobile nutrients such as phosphate (P), potassium (K+), and ammonium (NH4+) beyond the root zone and transfer phosphate from the fungus to the root cells. Thus, any process that was previously limited by the availability of phosphate will increase in rate. Improved phosphate nutrition, as well as direct fungal effects, may be implicated in the enhanced uptake of other macronutrients and micronutrients by plants.
Many VA mycorrhizal plants have increased resistance to disease compared to non-mycorrhizal controls, although the converse has also been reported. It has been suggested that physical protection of potential entry points on the root surface by the mycorrhizal fungi, and the reduced availability of carbon compounds to pests and pathogens (due to their being used by the fungi) are responsible for increased resistance to some diseases; however, the improved nutritional status of mycorrhizal plants made them more susceptible to other diseases (Pfleger and Linderman, 1994; Podila and Dodds,1999). A positive role for VA mycorrhizae in plant protection is indicated by the presence of phytoalexins (compounds involved in resistance to infection by pathogens), for example in mycorrhizal soybean roots (Morandi et al., 1984; Morandi, 1989; Harrison, 1999).
Certain changes in the plant hormonal balance have been shown to be related to a VA mycorrhizal effect. Some VA endomycorrhizae have been shown to contain elevated levels of phytohormones compared with non-infected roots. This may be due to the improved supply of nutrients, but could equally well be a direct result of the fungus, as it has been reported that plant growth promoting substances have been detected in germinating VA mycorrhizal fungal spores. Hormone accumulation in host tissue is affected by mycorrhizal infection, with changes in the levels of cytokinin, abscisic acid, and gibberellin-like substances and alteration in biomass partitioning (Barker et al., 1998; Danneberg et al., 1992; Dixon, 1992; Goicoechea, 1996).
The effect of VAM fungal infection on the drought resistance of plants is probably also due to the improvement of nutrient uptake. A mycorrhizal pathway of nutrient acquisition would become much more important in dry soil, because the nutrients become much less mobile. There seems little doubt that, like plant growth changes, mycorrhizal fungi can cause changes in plant water relations and may, at least in some cases, improve drought resistance of the plant (Al-Karaki et al., 1998; Goicoevhea et al., 1996; Nelson, 1987).
Morton and Benny (1990) described 149 species of VAM fungi. The VA mycorrhizal fungi are all classified as belonging to the family Endogonaceae possessing coenocytic hyphae (with only rare septa or none at all), which contain many nuclei. They have not propagated independently from host plant roots, and no sexual reproduction has observed, although both have been demonstrated in some non-mycorrhizal, saprophytic members of the Endogonaceae. Genera known to form VA mycorrhizae are Glomus, Gigaspora, Acaulospora, Sclerocystis, and Entrophospora. Knowledge of the phylogenetic relationships of VAM fungi is fragmentary. There is scant literature available on cytological or sexual processes involved in spore formation for these fungi. The taxonomic position of the VAM fungi is, therefore, inferred from developmental processes and spore morphology (Schenck and Yvonne, 1992; Morton, 1995).
The VA mycorrhizal fungi produce large vegetative spores (often greater than 100 xcexcm in diameter) usually on hyphae external to the roots. The genera have been classified according to the appearance of the spores. Spores may be recovered from the soil around infected plants by wet-sieving through a series sieves of different sizes, ranging from 1,000 xcexcm to 75 xcexcm. Either spores or fragments of infected roots may be used to inoculate further plants. Spores produced by Gigaspora, Acaulospora, and Entrophospora are called azygospores. Spores produced by Glomus and Sclerocystis, termed chlamydospores, are presumably asexual, thick-walled resting cells. Additional morphological characters that are useful in separating genera and species of VAM fungi are (i) the method of spore germination; (ii) presence or absence of sporocarps; (iii) presence or absence of auxiliary cells; (iv) spore dimensions; (v) spore color; (vi) spore ornamentation; (vii) number and type of spore walls; and (viii) histochemical reactions (Hall, 1984; Morton, 1988, 1995; Schenck and Yvonne, 1992).
Spores of several species of VAM fungi readily germinate on distilled water or semi-solid water agar, suggesting that nutritional requirements for germination are met by the mobilization of spore reserves. In some instances, an exogenous supply of nutrients may result in increased germination rates; however, no specific requirement for germination on water agar has been reported. The germination rates of spores of VAM fungi are improved by thiamine, nutrient broth medium, and soil extracts, root exudates, soil volatile compounds and small amounts of glucose (Carr et al., 1985; Carr et al., 1986; Elias and Safir, 1987; Graham, 1982; Hepper, 1979, 1983, 1984; Mosse, 1959; Smith and Gianinazzi-Pearson, 1988). Some early biochemical events in the germination of VAM fungal spores have been studied. A number of enzyme activities have been demonstrated in germinating spores: glutamate dehydrogenase suggesting amino acid respiration; succinate dehydrogenase suggesting the Krebs (TCA) cycle; glyceraldehyde-3-phosphate dehydrogenase indicating the Embden-Myerhof-Parnas glycolytic pathway; and glucose 6-phosphate dehydrogenase, which suggests the presence of the hexose monophosphate shunt. However, it is necessary to demonstrate all the relevant enzymes of these pathways to confirm their full metabolic functions (Beilby, 1982, 1983; Macdonald and Lewis, 1978). Acetate is incorporated into organic and amino acids, indicating that the TCA cycle and amino acid biosynthetic pathways are operative. Nuclear DNA synthesis has not been detected during germination, but nuclear division does occur (Burggraffand Beringer, 1989; Smith and Gianinazzi-Pearson, 1988).
In summary, spores of VAM fungi seem to be able to germinate readily, but not continuous growth and sporulation unless the fungal hyphae form symbiotic relationship with living roots. During root-fungus interactions, signal molecules from host plants are probably produced to turn on the fungal genes for growth and development. The symbiotic biology involved should be a focus of study in order to design artificial systems for cultivation of VAM fungi in large quantities (Smith and Read,1997; Hirsch and Kapulnik, 1998; Harrison, 1999).
Progress toward the culture of VAM fungi will be possible if systematic studies of the interactions between the fungi and their host plants are made. In this regard, biochemical and molecular events of the symbiosis between nitrogen fixing rhizobia and the roots of legume plants, the most understood system in detail, is highly instructive and relevant to the symbiosis between mycorrhizal fungi and host roots. In nitrogen fixation, expression of bacterial nod genes, which are essential to the initial step in the nodulation processes, is induced by root exudate. The active compounds in the exudates have been identified to be flavonoids or isoflavonoids (Long, 1989; Hirsch and Kapulnik, 1998; Vierheilig et al., 1998). Pea mutants, which do not form root nodules when inoculated with rhizobia, are designated as nod negative. The connection between symbiotic nitrogen fixation and mycorrhizal symbiosis has been established by the finding that certain nod pea mutants have been found to also be myc negative (inability to form vesicular-arbuscular mycorrhiza) (Duc et al., 1989). This discovery suggests that several early steps in the processes of forming nodules on one hand and mycorrhizae on the other may involve common plant functions. To test the hypothesis that root exudates and flavonoids may be signal molecules in the early interactions between the fungi and the host plants, the effect of these bioactive chemicals on spore germination and rate of hyphae growths were investigated. Both root exudates and selected flavonoids significantly increased the frequency of spore germination and the rate of hyphal growth. The sensitivity of VAM fungi to small amounts (0.5 to 1.5 xcexcM) of bioactive molecules suggests that flavonoids may acts as signals to induce VAM fungal development in the early stages of the symbiosis (Gianinazzi-Pearson et al., 1989; Hirsch and Kapulnik, 1998; Harrison, 1999).
VAM inocula are likely to play an increasingly important role in agriculture because their presence would not only reduce the use of applied fertilizers and pesticides but would also increase the resistance of plants to environmental and biological stresses (Cooper, 1987, Dehn, 1987, Hayman, 1987, Nelson, 1987). Current supplies of fungal inocula for experimental work are produced in pot cultures containing soil and whole plants. The resulting spores and propagules are contaminated with a variety of adventitious microorganisms.
Cultivation of VAM fungi under axenic conditions has been attempted without success for many years (Smith and Gianinazzi-Pearson, 1988). Production of VAM fungi on an industrial scale has not been feasible up to the present time because practical methods of cultivating VAM fungi have not been discovered. This is one of the key challenges for utilization of VAM fungi.
Hairy root organ culture of carrot, produced by transforming carrot slices of Ri plasmid (Ri, root inducing) of Agrobacterium rhizogenes, can be easily propagated and maintained. Hence, it may be used to monitor interaction of roots with VAM fungi. Becard and Fortin (1988) demonstrated that infectious spores of Gigaspora margarita were produced in hairy root tissue culture. Mugnier and Mosse (1987) have successfully used Ri Txe2x80x94DNA transformed morning glory roots as host for the colonization of VAM fungi. The hairy roots may provide an ideal system for supporting the cultivation of VAM fungi.
Mugnier (U.S. Pat. No. 4,599,312) discloses a method of producing endomycorrhizal fungi with vesicles and arbuscules in vitro by producing dicotyledonous roots that have been genetically converted by inserting genes of root-inducing or tumor-inducing plasmid into the genome of dicotyledonous roots, and then inoculating the transformed roots with endomycorrhizal spores.
Fortin (U.S. Pat. No. 5,554,530) discloses an aseptic method of producing VAM propagules by infecting transformed roots in vitro in a two-compartment container.
Sylvia et al. (U.S. Pat. No. 5,096,481) discloses a VAM inoculum composition comprising host root plants colonized by a least one species of VAM, the colonized roots having a particle size in the range of from about 33 xcexcm to about 425 xcexcm and a propagule density of up to about 1,000,000 VAM fungal propagules per gram dry mass of host plant root and method for encapsulating the composition and methods for enhancing plant growth utilizing the inocula. The method taught by Sylvia et al. is not aseptic as the method of the present invention.
Wood et al. (EP-209,627) disclose a method of producing fungal inoculum grown in axenic root organ cultures produced in porous substrates wetted with solutions. This procedure produces a small number of spores in the order of 83 spores per 30 ml of medium.
Okii (U.S. Pat. No. 4,945,059) disclose a method of proliferating VAM spores using potato roots and a porous amphoteric ion exchanger. This method is not aseptic as the method of the present invention.
Mosse et al. (U.S. Pat. No. 4,294,037) disclose a method of producing VAM fungi on plant roots in nutrient film culture. The technique requires continuous re-cycling of a large volume of nutrient liquid in a film which flows over the roots of plants. The present invention does not require large volumes of nutrient media for the production of VAM.
Several patents disclose various methods of dispersing VAM. Tuse et al. (U.S. Pat. No. 5,344,471) teach coating compositions comprising a polymer, VAM propagules, and a fungicide that selectively inhibits pathogenic fungi and several methods of coating roots with such compositions. Warner (U.S. Pat. No. 4,551,165) describes seed pellets made of a mixture of peat, a binder, seed, and VAM. Janerette (U.S. Pat. No. 5,178,642) teaches fungal inoculants made of a particulate carrier and a nutrient solution.
There is not disclosed a method for a continuous, aseptic in vitro production of VAM, Glomus intraradices, in particular.
The present invention is directed to novel fungal growth media.
The present invention shows the successful mass production of biologically active spores of vesicular arbuscular mycorrhizae (VAM) in an axenic transformed dicotyledonous root organ culture.
An object of the invention is to provide a method for the in vitro mass production of VAM, mainly spores.
Another object of the invention is to provide a method for the in vitro mass production of Glomus intraradices, mainly spores.
A further object of the present invention is to provide for the in vitro aseptic mass production of VAM (Glomus intraradices, in particular), mainly spores, which are contamination-free, simple, inexpensive, and effective.
Another object of the invention is to provide for axenic growth of continuous cultures of VAM (Glomus intraradices, in particular).
Still another object of the invention is to provide for conditions that will promote spore production of VAM (Glomus intraradices).
A further object of the invention is to provide for compositions comprising biologically pure cultures of VAM (Glomus intraradices, in particular), propagated by the methods of the invention. The composition may comprise a biodegradable material suitable as a carrier in the composition, including peat moss, vermiculite, perlite, alginate polymer, and xanthan-chitosan complex etc. These compositions may also comprise isoflavonoids, alkali formononetinate, or both.
The invention provides for a method of proliferating VAM (Glomus intraradices, in particular) by inoculating the VAM fungi directly on or around the roots of plants including: citrus, such as orange; fruits, such as strawberry; ornamental plants, such as rose; fruit and nut trees; specialty crops, such as ginseng; artificial seeds such as alfalfa embryos; crops, such as soybean, wheat, and corn; and plantlets grown from tissue culture.